Smelly shark, smelly ray: what is infecting you?

AIMS: Although elasmobranchs are consumed worldwide, bacteriological assessments for this group are still sorely lacking. In this context, this study assessed bacteria of sharks and rays from one of the most important landing ports along the Rio de Janeiro coast. METHODS AND RESULTS: Bacteria were isolated from the cloacal swabs of the sampled elasmobranchs. They were cultured, and Vibrio, Aeromonas, and Enterobacterales were isolated and identified. The isolated bacteria were then biochemically identified and antimicrobial susceptibility assays were performed. Antigenic characterizations were performed for Salmonella spp. and Polymerase Chain Reaction (PCR) assays were performed to identify Escherichia coli pathotypes. Several bacteria of interest in the One Health context were detected. The most prevalent Enterobacterales were Morganella morganii and Citrobacter freundii, while Vibrio harveyi and Vibrio fluvialis were the most prevalent among Vibrio spp. and Aeromonas allosacharophila and Aeromonas veronii bv. veronii were the most frequent among Aeromonas spp. Several bacteria also displayed antimicrobial resistance, indicative of Public Health concerns. A total of 10% of Vibrio strains were resistant to trimethoprim-sulfamethoxazole and 40% displayed intermediate resistance to cefoxitin. Salmonella enterica strains displayed intermediate resistance to ciprofloxacin, nalidixic acid and streptomycin. All V. cholerae strains were identified as non-O1/non-O139. The detected E. coli strains did not exhibit pathogenicity genes. This is the first study to perform serology assessments for S. enterica subsp. enterica isolated from elasmobranchs, identifying the zoonotic Typhimurium serovar. Salmonella serology evaluations are, therefore, paramount to identify the importance of elasmobranchs in the epidemiological salmonellosis chain. CONCLUSIONS: The detection of several pathogenic and antibiotic-resistant bacteria may pose significant Public Health risks in Brazil, due to high elasmobranch consumption rates, indicating the urgent need for further bacteriological assessments in this group.

Identification and characterization of motile Aeromonas spp. isolated from farmed Nile tilapia (Oreochromis niloticus) in the Philippines.

AIMS: Motile Aeromonas septicaemia (MAS) caused by motile Aeromonas species is an important disease in farmed freshwater fish due to intensification of culture and improper farm practices. This study characterized and profiled motile Aeromonas species recovered from clinically sick tilapia farmed in the Philippines, with a view to identifying targeted disease prevention and control measures against MAS in farmed tilapia species. METHODS AND RESULTS: Sixteen isolates from diseased farmed Nile tilapia were identified as Aeromonas veronii (n = 14), Aeromonas caviae (n = 1), and Aeromonas dhakensis (n = 1). Five biochemical profiles using API 20E were exhibited by the A. veronii strains giving an unreliable identification. A high level of agreement was observed in identifying the Aeromonas strains using 16S rRNA and rpoD gene sequencing, although the latter has a higher discriminatory value. Three or more virulence genes dominated by cytotoxic enterotoxin act and aerolysin aer were detected. Different genotypes based on virulence gene clustering suggested varied mechanisms used by Aeromonas to colonize and infect or to mutualistically co-exist with the fish. Acquired multiple antibiotic resistance was found in a single A. veronii isolate. All were susceptible to enrofloxacin, oxolinic acid, florfenicol, and chloramphenicol. Tetracycline and sulfonamide resistances and class 1 integron were detected in three A. veronii isolates. CONCLUSION: Several strains of motile aeromonads, especially A. veronii, which have varied genotypes based on virulence, biochemical profile, and antibiotic resistance, are involved in MAS in natural disease outbreaks in farmed Nile tilapia in the Philippines.

Occurrence of potential virulence determinants in Aeromonas spp. isolated from different aquatic environments.

Aeromonas species are bacterial pathogens that cause significant economic losses in the aquaculture industry worldwide. They are widely distributed in aquatic environments and cause several diseases in both humans and aquatic animals. The presence of various virulent Aeromonas spp. in aquatic environments predisposes infections in aquatic animals and humans. Concern about the transfer of pathogens from fish to humans also increased with the substantial increase in seafood consumption. Aeromonas spp. are also primary human pathogens that cause local and systemic infections, both in immunologically compromised and immunologically competent hosts. The most common Aeromonas spp. causing infections in aquatic animals and humans are A. hydrophila, A. salmonicida, A. caviae, and A. veronii biotype sobria. The ability of Aeromonas spp. to produce a variety of virulence factors enhances their pathogenic ability. Literature has supplied evidence for the presence of various virulence factors, including proteases, enterotoxins, hemolysin, and toxin genes of Aeromonas spp. in aquatic environments. The high prevalence of Aeromonas spp. in the aquatic environment is also a threat to public health. Because the Aeromonas spp. infections in humans are generally the result of ingestion or exposure to contaminated food and water. This review summarizes the recently published information on various virulence factors and virulence genes of Aeromonas spp. isolated from various aquatic environments, including seawater, freshwater, wastewater, and drinking water. It is also intended to highlight the risks associated with Aeromonas species' virulence properties for both aquaculture and public health.

Evaluation of three Bacillus spp. isolated from the gut of giant freshwater prawn as potential probiotics against pathogens causing Vibriosis and Aeromonosis.

Pathogens from the Vibrio and Aeromonas genera often cause detrimental effects to the aquaculture sector. Previously, antibiotics was used to resolve the infections, but this caused the spread of antibiotic resistant bacteria and antibiotic resistance genes into the environment. As an approach to address this issue, probiotic bacteria were introduced to improve the hosts' microbiome, disease protection, health condition, growth efficiency, feed consumption, stress response and general vigour. However, reports showed that some commercially available probiotics were restricted to a small number of microbial species and there are inconsistencies concerning its effectiveness. Hence, the aim of this study was to isolate and evaluate new Bacillus spp. from the gut of giant freshwater prawn as potential probiotics. Three Bacillus spp. isolates, Bacillus subtilis FS6 (MZ960135), Bacillus velezensis FS26 (MZ960133) and Bacillus pumilus FS97 (MZ960136) were characterised, and in vitro testing showed good probiotic properties which can help in dealing with diseases in aquaculture. Among the Bacillus spp., Bacillus velezensis FS26 showed higher antimicrobial activity towards Aeromonas hydrophila LMG 13658 and Aeromonas veronii clone DK-A. veronii-27 at 23.7 mm and 25 mm, respectively. Bacillus subtilis FS6 and Bacillus velezensis FS26 resulted in good adherence to both xylene and chloroform hydrocarbons. The Bacillus spp. isolated displayed high survivability towards 0.3% bile salt and exhibited amylase, protease, and lipase activities. Thus, the isolated Bacillus spp. are considered safe based on the sensitivity analysis towards antibiotics and γ-haemolytic activity.

Diversity and antimicrobial susceptibility profiles of Aeromonas spp. isolated from diseased freshwater fishes in Thailand.

Motile Aeromonas septicemia (MAS), a disease caused by Aeromonas spp., is recognized as a major disease in freshwater aquaculture. This study aimed to investigate the distribution and diversity of Aeromonas spp. and their antimicrobial susceptibility patterns. A total of 86 isolates of Aeromonas spp. were recovered from diseased freshwater fishes from 13 farms in Thailand. All isolates were identified using biochemical characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), polymerase chain reaction assays, and the gyrB gene sequence analysis. The result of MALDI-TOF MS showed 100% (86 isolates) accuracy at genus-level identification, and 88.4% (76 isolates) accuracy at species-level identification. Six species of Aeromonas were confirmed through nucleotide sequencing and phylogenetic analysis of the gyrB gene Aeromonas veronii (72.1%), Aeromonas jandaei (11.6%), Aeromonas schubertii (9.3%), Aeromonas diversa (3.5%), Aeromonas hydrophila (2.3%), and Aeromonas punctata (1.2%). Antimicrobial susceptibility tests for all isolates revealed resistance against amoxicillin (99%), ampicillin (98%), oxolinic acid (81.4%), oxytetracycline (77%), trimethoprim-sulfamethoxazole (24%), and enrofloxacin (21%). The multiple antibiotic resistance (MAR) index varied between 0.14 and 0.86, with MAR values more than 0.2 in 99% of isolates. Furthermore, four diverse multidrug-resistant (MDR) patterns were found among Aeromonas isolates. Our finding show that A. veronii is the most abundant species in Thai cultured freshwater fish with the highest MDR patterns.

Delivering the pain: an overview of the type III secretion system with special consideration for aquatic pathogens.

Gram-negative bacteria are known to subvert eukaryotic cell physiological mechanisms using a wide array of virulence factors, among which the type three-secretion system (T3SS) is often one of the most important. The T3SS constitutes a needle-like apparatus that the bacterium uses to inject a diverse set of effector proteins directly into the cytoplasm of the host cells where they can hamper the host cellular machinery for a variety of purposes. While the structure of the T3SS is somewhat conserved and well described, effector proteins are much more diverse and specific for each pathogen. The T3SS can remodel the cytoskeleton integrity to promote intracellular invasion, as well as silence specific eukaryotic cell signals, notably to hinder or elude the immune response and cause apoptosis. This is also the case in aquatic bacterial pathogens where the T3SS can often play a central role in the establishment of disease, although it remains understudied in several species of important fish pathogens, notably in Yersinia ruckeri. In the present review, we summarise what is known of the T3SS, with a special focus on aquatic pathogens and suggest some possible avenues for research including the potential to target the T3SS for the development of new anti-virulence drugs.

Aeromonas spp. isolated from ready-to-eat seafood on the Norwegian market: prevalence, putative virulence factors and antimicrobial resistance.

AIMS: We aim to investigate the prevalence, putative virulence factors and antimicrobial resistance of mesophilic Aeromonas isolated from ready-to-eat (RTE) seafood available on the Norwegian market, and to assess the potential risks by consuming RTE seafood to consumers. METHODS AND RESULTS: The prevalence of mesophilic Aeromonas in 148 RTE seafood was investigated and the highest prevalence was found in retail sushi (17%), followed by oysters (10%), fresh salmon loins (10%) and scallops (4%). Among 43 Aeromonas isolates, 75% of them were identified as A. media, 23% as A. salmonicida and 2% as A. bestiarum based on partial gryB gene sequencing. Aeromonas isolates were potentially pathogenic due to the presence of four virulence genes: alt (73%), hylA (22%), aerA (17%) and act (6%). In addition, all isolates were resistant to ampicillin and erythromycin. Most of the isolates (98%) were multidrug resistant. CONCLUSIONS: The occurrence of potentially pathogenic and multidrug-resistant Aeromonas strains in RTE seafood implies a potential risk to consumers. Our finding suggests that RTE seafood could be a potential vehicle for the transfer of virulent and multidrug-resistant Aeromonas. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study to report multiple antibiotic resistance in Aeromonas associated with RTE seafood in Norway.

Virulence and antimicrobial resistance potential of Aeromonas spp. associated with shellfish.

Aeromonas spp. are associated with seafood-related outbreaks worldwide. In seafood industry, shellfish play a major role in global seafood production. With this emerging trend of shellfish consumption, shellfish-related bacterial infections are being reported frequently. Aeromonas spp. are natural contaminants found in shellfish. Although 36 species have been identified, some species including Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii biotype sobria have dragged major attention as foodborne pathogenic bacteria. The ability to elaborate a variety of virulence factors of Aeromonas spp. contributes to the pathogenic activities. Also, emerging antimicrobial resistance in Aeromonas spp. has become a huge challenge in seafood industry. Furthermore, multidrug resistance increases the risk of consumer health. Studies have supplied pieces of evidence about the emerging health risk of Aeromonas spp. isolated from seafood. Therefore, the present review was intended to highlight the prevalence, virulence and antimicrobial resistance of Aeromonas spp. isolated from various types of shellfish.

Ornamental fish: a potential source of pathogenic and multidrug-resistant motile Aeromonas spp.

Aeromonas spp. are ubiquitous bacteria that cause diseases in fish and other aquatic animals. They are the natural inhabitants of different aquatic environments, such as freshwater, brackishwater and marinewater. Extrinsic stressors, such as crowding, unhygienic handling, poor water quality, polluted feeding and inadequate nutrition, can predispose fish to Aeromonas infection. In ornamental fish, motile Aeromonas spp. are known as aetiological agents of motile aeromonad infections, which cause significant mortality in fish and economic loss in the ornamental fish industry. The existence of different virulence factors leads to the virulence potential of motile Aeromonas spp. There are several antimicrobials used to treat bacterial infections in ornamental fish. However, the extensive use of antimicrobials in the ornamental fish industry causes multidrug resistance. This article reviewed a multitude of virulence factors that are related to the ornamental fish-borne Aeromonas pathogenicity and the antimicrobial resistance determinants related to the multidrug resistance phenotypes of motile Aeromonas spp. in ornamental fish.

Clinical and microbiological characteristics of Aeromonas bacteremia in Turkey.

We investigated the cases with Aeromonas bacteremia in terms of clinical and microbiological characteristics, underlying disease and mortality rates. Patients with positive blood cultures were included in this research. Aeromonas bacteremia was diagnosed as at least one positive blood culture for Aeromonas species. The bacteremia was defined as community origin if the onset was in the community or within 72 hours of hospital admission. The others were considered as nosocomial. All bacteria were defined as Aeromonas with conventional method. Species identification was verified by VITEK system. Antibiotic susceptibility tests were analyzed with the disc diffusion, E-test method or VITEK system. Thirty-three patients were diagnosed with bacteremia due to Aeromonas spp. Hematologic and solid tumors were the leading underlying conditions, followed by cirrhosis. Two patients (6%) had community-acquired infections. Aeromonas hydrophila was the most common isolated bacterium. The crude mortality rate was 36%. 12 patients died and 6 deaths and 4 deaths were detected in patients with bacteremia caused by A. hydrophila and Aeromonas sobria respectively. All strains were resistant to ampicillin and more than 90% of the strains were susceptible to trimethoprim-sulfamethoxazole, fluoroquinolone, third generation cephalosporins, and carbapenems. Aeromonas sp. is not a frequent cause of bacteremia however, it may lead to high mortality rates, especially in the immunocompromised hosts and patients with liver cirrhosis. Nosocomial Aeromonas bacteremia is not uncommon in these populations. Broad-spectrum cephalosporins, piperacillin-tazobactam, fluoroquinolones, and carbapenems remain as effective antimicrobial agents for therapy of Aeromonas bacteremia.

Fatty Acids-Enriched Fractions of Hermetia illucens (Black Soldier Fly) Larvae Fat Can Combat MDR Pathogenic Fish Bacteria Aeromonas spp.

Aeromonas spp. cause many diseases in aquaculture habitats. Hermetia illucens (Hi) larvae were used as feed-in aquacultures and in eradicating pathogenic fish bacteria. In the present study, we applied consecutive extractions of the same biomass of BSFL fat using the acidic water-methanol solution. The major constituents of the sequential extracts (SEs) were free fatty acids (FFAs), and fatty acids derivatives as identified by gas chromatography spectrometry (GC-MS). Our improved procedure enabled gradual enrichment in the unsaturated fatty acids (USFAs) content in our SEs. The present study aimed to compare the composition and antimicrobial properties of SEs. Among actual fish pathogens, A. hydrophila and A. salmonicida demonstrated multiple drug resistance (MDR) against different recommended standard antibiotics: A. salmonicida was resistant to six, while A. hydrophila was resistant to four antibiotics from ten used in the present study. For the first time, we demonstrated the high dose-dependent antibacterial activity of each SE against Aeromonas spp., especially MDR A. salmonicida. The bacteriostatic and bactericidal (MIC/MBC) activity of SEs was significantly enhanced through the sequential extractions. The third sequential extract (AWME3) possessed the highest activity against Aeromonas spp.: inhibition zone diameters were in the range (21.47 ± 0.14-20.83 ± 0.22 mm) at a concentration of 40 mg/mL, MIC values ranged between 0.09 and 0.38 mg/mL for A. hydrophila and A. salmonicida, respectively. AWME3 MBC values recorded 0.19 and 0.38 mg/mL, while MIC50 values were 0.065 ± 0.004 and 0.22 ± 0.005 mg/mL against A. hydrophila and A. salmonicida, respectively. Thus, the larvae fat from Hermitia illucens may serve as an excellent reservoir of bioactive molecules with good capacity to eradicate the multidrug-resistant bacteria, having promising potential for practical application in the aquaculture field.

Phylogenetic characteristics, virulence properties and antibiogram profile of motile Aeromonas spp. isolated from ornamental guppy (Poecilia reticulata).

Aeromonas spp. are opportunistic pathogenic bacteria related to an assembly of infectious diseases in ornamental fish. In the present study, virulence properties and antibiotic susceptibility of 52 guppy-borne Aeromonas spp. were investigated. The isolates were identified as A. veronii (n = 34), A. dhakensis (n = 10), A. hydrophila (n = 3), A. caviae (n = 3) and A. enteropelogenes (n = 2) by gyrB gene sequencing. The gyrB sequence deviation within and among the species ranged from 0 to 2.6% and 2.7-9.2%. Each species formed a distinct group in the unrooted neighbor-joining phylogenetic tree. The phenotypic virulence factors such as ß-hemolysis, slime, caseinase, DNase, gelatinase and lipase production were observed in 28 (53.9%), 33 (63.5%), 28 (53.9%), 42 (80.8%), 37 (71.2%) and 42 (80.8%) isolates, respectively. The virulence genes were detected by PCR assay in the following proportions- act (84.6%), hly (80.8%), aer (73.1%), lip (73.1%), gcaT (73.1%), ascV (53.8%), ahyB (53.8%) fla (51.9%), alt (48.1%), ast (36.5%) and ser (34.6%), respectively. The amoxicillin, ampicillin, imipenem, nalidixic acid, oxytetracycline and rifampicin were resistant to more than 70.0% of the isolates in antibiotic susceptibility test. Our study suggests that the ornamental guppy can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp.

Antimicrobial resistance of pathogenic Aeromonas spp. isolated from marketed Pacific abalone (Haliotis discus hannai) in Korea.

AIMS: The object of this study was to identify potential health concerns of the Aeromons spp. isolated from marketed Pacific abalone (Haliotis discus hannai) with respect to their virulence and antimicrobial resistance patterns. METHODS AND RESULTS: We identified 29 strains of aeromonads consisting of five species; Aeromonas hydrophila (n = 9), Aeromonas enteropelogenes (n = 14), Aeromonas veronii (n = 3), Aeromonas salmonicida (n = 2) and Aeromonas sobria (n = 1), by employing series of biochemical tests and gene sequencing. In the phenotypic virulence assays, all isolates showed gelatinase and caseinase activities, while lipase formation (69%), phospholipase production (90%), DNase formation (82%), slime production (49%) and haemolysis activity (α = 18% and ß = 82%) were also detected among isolates. Prevalence of virulence genes; aerA (100%), fla (66%), ahyB (73%), act (52%), alt (42%), ast (35%), ser (52%), gcat (69%), ascV (43%), hlyA (83%), lip (52%) and exu (59%) were detected by PCR assays. In disc diffusion test, 100% resistance was detected against ampicillin while cephalothin, rifampicin, oxytetracycline, colistine sulphate, nalidixic acid and piperaciliin were resisted by 86, 73, 42, 35, 28, 20 and 20% of the isolates respectively. Thirteen (45%) of the isolates showed multiple antimicrobial resistance (MAR) indices ≥ 0·2. CONCLUSIONS: Our findings suggest that the potential health risk posed by the abalone-borne Aeromonas spp. should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time to evaluate possible public health risks upon consumption of abalone harbored Aeromonas spp. and also to isolate potential pathogenic and multidrug-resistant Aeromonas spp. from Pacific abalone in Korea.

Prevalence of virulence and antimicrobial resistance genes in Aeromonas species isolated from marketed cockles (Tegillarca granosa) in Korea.

This study aimed to examine incidence, virulence and antimicrobial properties in Aeromonas spp. isolated from cockles (Tegillarca granosa) in Korea. Firstly, genomic DNA was extracted from 32 Aeromonas spp. isolates, and PCR screening for virulence, antimicrobial resistance genes was carried out. The disk diffusion assay was used to examine antimicrobial susceptibility. Aeromonas spp. isolates comprised, A. hydrophila (n = 8), A. veronii (n = 15), A. media (n = 2), A. salmonicida (n = 2), A. allosaccharophila (n = 1), A. bestiarum (n = 1), A. culicicola (n = 1), A. enteropelogenes (n = 1) and A. rivipollensis (n = 1). High prevalence of virulence-related genes reported as; act (69%), alt (47%), ast (41%), aerA (56%), lip (50%), ahyB (47%), ser (28%), fla (66%), gcat (44%), ascV (50%) and hlyA (72%). All isolates were multidrug resistant, while highest resistance level observed for ampicillin (100%), followed by imipenem (81%), rifampicin (78%), cephalothin (72%), piperacillin (47%) and Colistin sulfate (31%). The presence of blaSHV , blaCTX , tetE, aac(6')-Ib, strA-strB, qnrS, qnrB and IntI1 genes were reported in varying combinations. Nevertheless, blaTEM , blaIMP , tetA, tetB, qnrA, qnrB and aphAI-IAB genes and the class1 integron were not detected. The high occurrence of virulence and antimicrobial resistance genes in cockles reveals that it can be a potential health risk source for consumers.

Characterization of co-culture of Aeromonas and Pseudomonas bacterial biofilm and spoilage potential on refrigerated grass carp (Ctenopharyngodon idellus).

Aeromonas and Pseudomonas are important bacterial species involved in spoilage of refrigerated freshwater fish. In this study, 10 Aeromonas and seven Pseudomonas bacterial strains were isolated from spoiled grass carp and identified. Twelve of seventeen bacterial strains showed high potential of biofilm formation and 14 of 17 can produce extracellular protease. In order to explore the spoilage capacity of dual-species, the sterile grass carp fillets were inoculated with mono- and dual-species of Aeromonas salmonicida and Pseudomonas azotoformans strains. The results revealed significantly higher levels of the total viable count and total volatile basic nitrogen in dual-species as compared to mono-species from day 6. The higher contents of histamine, cadaverine and serious degradation in muscles tissue were also observed in dual-species after 10 days of storage. Results of in vitro experiments showed that the co-culture of A. salmonicida and P. azotoformans significantly increased the bacterial maximum growth rate, promoted the biofilm formation and improved the spoilage capacity of bacterial strains. This study has revealed that the co-culture of Aeromonas and Pseudomonas bacterial strains accelerated spoilage process of grass carp and increased biofilm formation. It indicates that the mixed-cultures of spoilage micro-organisms pose a huge threat to food industry.

The determination of antimicrobial susceptibility by MIC and epidemiological cut-off values and the detection of resistance genes in Aeromonas species isolated from cultured fish.

The present study was aimed at determining antimicrobial susceptibility by a CLSI standard microdilution testing protocol and detecting the resistance genes of motile Aeromonas species isolated from cultured fish. The importance of the minimum inhibitory concentrations was assessed based on statistically determined epidemiological cut-off values calculated by normalized resistance analysis. Unfortunately, CLSI epidemiological cut-off values are available only for Aeromonas salmonicida, and there is no further detailed data on Aeromonas isolated from aquatic animals. The antimicrobial susceptibilities of pre-identified motile Aeromonas species to florfenicol, tetracycline and sulfamethoxazole were determined by calculating epidemiological cut-off values with fully automated and freely available Excel spreadsheets, applying the normalized resistance interpretation (NRI) method. Furthermore, the presence of the antimicrobial resistance genes floR, tetA, tetB, tetC, tetD, tetE, tetH, sulI, sulII and sulIII was detected by PCR analysis and confirmed by sequence analysis. The presence of up to six different genes (multiple antimicrobial resistance) was determined in the Aeromonas isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: In this study, we investigated phenotypic and genotypic antimicrobial resistance characteristics by a novel method based on epidemiological cut-off values. This is the second comprehensive study on the antimicrobial susceptibility characteristics of Aeromonas species using NRI and epidemiological cut-off values. The present research is related to our previous researches focussed on the identification of motile Aeromonads, their prevalence in relation to different fish lengths, seasons and regions, and covered the investigation of Lactococcus garvieae, Yersinia ruckeri, Flavobacterium spp., Enterobacter spp. and Citrobacter spp.

Aeromonas spp. from marketed Yesso scallop (Patinopecten yessoensis): molecular characterization, phylogenetic analysis, virulence properties and antimicrobial susceptibility.

AIMS: Yesso scallop (Patinopecten yessoensis) is a popular seafood in Korea. Aeromonas spp., well-known pathogenic bacteria, has been reported in some molluscan shellfish, but it has not been studied in scallops so far. Therefore, we aimed to isolate, identify and characterize the Aeromonas spp. isolated from marketed Yesso scallops to estimate their potential risk to public health. METHODS AND RESULTS: Thirty-two Aeromonas spp. including A. hydrophila (n = 13), A. salmonicida (n = 11), A. media (n = 3), A. caviae (n = 2), A. veronii (n = 2) and A. enteropelogenes (n = 1) were isolated from 105 marketed scallops and tested for phenotypic pathogenicity, virulence genes and antimicrobial susceptibility. Mean total bacterial count of scallop meat was 1·34 × 104 CFU per gram. Slime production and lipase tests were positive in 97% of the isolates while DNase, protease, gelatinase, phospholipase and haemolysis were shown by 88, 88, 81, 88 and 72% of the isolates respectively. Eleven virulence genes were detected among Aeromonas spp. (act (75%), alt (59%), ast (47%), aerA (78%), lip (59%), ahyB (94%), ser (75%), hlyA (75%), fla (64%), gcat (84%) and ascV (23%)), and exu was negative in all isolates. Aeromonas hydrophila and A. salmonicida harboured ≥7 virulence genes and positive for enterotoxin genes, act, alt and ast. All the isolates were multidrug resistant and 100% resistant to ampicillin, colistin, vancomycin and cephalothin. Also, 30, 31, 20, 21, 29, 24, 27 and 27 of the isolates were resistant to piperacillin, clindamycin, erythromycin, nalidixic acid, imipenem, meropenem, trimethoprim-sulfamethoxazole and rifampicin respectively. CONCLUSIONS: It is obvious with our results that the Aeromonas spp. isolated from Yesso scallops are highly virulent and potentially pathogenic, whereas the multidrug resistance further expedite their importance. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study reporting Aeromonas spp. in scallop. This implies that not only the common varieties like oysters, but other bivalves can also harbour potentially pathogenic aeromonads which may have impacts on consumer health.

Antibiotic and heavy metal resistance genes in Aeromonas spp. isolated from marketed Manila Clam (Ruditapes philippinarum) in Korea.

AIMS: Manila clam (Ruditapes philippinarum) is one of the most popular seafood in Korea, owing to their unique taste and nutritional value. This study aimed to disclose the antibiotic and heavy metal resistance characteristics of Aeromonas spp. isolated from marketed Manila clam in Korea. METHODS AND RESULTS: A total of 36 Aeromonas spp. strains were isolated and subjected to two tests: an antibiotic disk diffusion test to determine their resistance to antibiotics, and a broth dilution test to determine their resistance to heavy metals. PCR-based amplification was performed to detect the resistance genes. A high level of resistance to ampicillin (100%) and cephalothin (89%) was observed, while 42, 39, 36 and 36% of the isolates were resistant to oxytetracycline, imipenem, nalidixic acid and tetracycline respectively. In addition, among the tested heavy metals, cadmium (Cd) recorded the highest resistance rate (61%), followed by chromium (Cr) (50%), lead (Pb) (47%) and copper (Cu) (37%). However, mercury (Hg) resistance was not observed. PCRs revealed the occurrence of blaTEM , blaSHV , blaCTX-M , qnrS, tetB, tetE, aac(6')-Ib, strA-strB and intI1 genes among 100, 31, 31, 78, 78, 89, 25, 50 and 72% of the isolates respectively. Moreover, heavy metal resistance genes, copA, merA and czcA were detected in 25, 47 and 61% of the isolates respectively. CONCLUSIONS: The results suggest the importance of multi-drug and heavy metal-resistant aeromonads in Manila clam to assess the consumer safety and public health. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to elaborate on the importance of multi-drug and heavy metal-resistant aeromonads in Manila clam. Particularly, the presence of extended-spectrum-ß-lactamase genes and other antibiotic resistance genes intensifies the possible health risks and may complicate therapeutic treatments upon infection, while heavy metal resistance suggests possible heavy metal exposure.

Multidrug resistant Aeromonas spp. isolated from zebrafish (Danio rerio): antibiogram, antimicrobial resistance genes and class 1 integron gene cassettes.

Aeromonas spp. are Gram-negative opportunistic bacteria which have been commonly associated with fish diseases. In this study, antibiogram, antimicrobial resistance genes and integrons of 43 zebrafish-borne Aeromonas spp. were studied. The isolates were identified as six Aeromonas species (A. veronii biovar veronii (n = 26), A. veronii biovar sobria (n = 3), A. hydrophila (n = 8), A. caviae (n = 3), A. enteropelogenes (n = 2) and A. dhakensis (n = 1)). Antibiogram of the isolates indicated that most of them were resistant to amoxicillin (100·00%), nalidixic acid (100·00%), oxytetracycline (100·00%), ampicillin (93·02%), tetracycline (74·42%), rifampicin (67·44%) and imipenem (65·15%). Multiple antimicrobial resistance (MAR) index values ranged from 0·19-0·44 to 90·70% isolates showed multidrug resistance. PCR of antimicrobial resistance genes revealed that the tetracycline resistance gene (tetA) was the most predominant (67·44%) among the isolates. The qnrS (53·49%), tetB (30·23%), tetE (30·23%), qnrB (23·26%) and aac(6')-Ib-cr (4·65%) genes were also detected. Class 1 integrase (IntI1) gene was found in 46·51% of the isolates. Two types of class 1 integron gene cassette profiles (qacG-aadA6-qacG and drfA1) were identified. The results showed that zebrafish-borne aeromonads can harbour different types of antimicrobial resistance genes and class 1 integrons. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas spp. are important pathogens found in diverse environments. Antimicrobial resistance genes and integrons of ornamental fish-borne Aeromonas spp. are not well studied. The antibiogram, antimicrobial resistance genes and class 1 integrons of Aeromonas spp. isolated from zebrafish were characterized for the first time in Korea. The prevalence of tetracycline resistance genes, plasmid-mediated quinolone resistance genes and class 1 integron gene cassettes were observed among the isolates. The qacG-aadA6-qacG gene cassette was identified for the first time in Aeromonas spp. The results suggest that the wise use of antimicrobials is necessary for the better management of the ornamental fish.

Diagnostic methods for identifying different Aeromonas species and examining their pathogenicity factors, their correlation to cytotoxicity and adherence to fish mucus.

Aeromonas spp. are ubiquitous in the aquatic environment, acting as facultative or obligate pathogens for fish. Identifying Aeromonas spp. is important for pathogenesis and prognosis in diagnostic cases but can be difficult because of their close relationship. Forty-four already characterized isolates of Aeromonas spp. were analysed by 16S rRNA gene sequencing, by gyrase B sequencing, by analysing their fatty acid profiles, by biochemical reactions and by MALDI-TOF MS. To determine their pathogenicity, cytotoxicity, adhesion to mucus and the expression of 12 virulence factors were tested. The susceptibility of the isolates towards 13 different antibiotics was determined. MALDI-TOF MS was found to be an acceptable identification method for Aeromonas spp. Although the method does not detect all species correctly, it is time-effective and entails relatively low costs and no other methods achieved better results. A high prevalence of virulence-related gene fragments was detected in almost all examined Aeromonas spp., especially in A. hydrophila and A. salmonicida, and most isolates exhibited a cytotoxic effect. Single isolates of A. hydrophila and A. salmonicida showed multiple resistance to antibiotics. These results might indicate the potentially pathogenic capacity of Aeromonas spp., suggesting a risk for aquatic animals and even humans, given their ubiquitous nature.